This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

Abstract

Background

To establish a model of pancreatic cancer induced by 7,12-dimethylbenzantracene (DMBA)
in Sprague–Dawley (SD) rats, and detect the expression of DNA-repair proteins (MGMT,
ERCC1, hMSH2, and hMLH1) and their significance in pancreatic cancer and non-cancerous pancreatic tissues
of SD rats.

Methods

DMBA was directly implanted into the parenchyma of rat pancreas (group A and group
B), and group B rats were then treated with trichostatin A (TSA). The rats in both
groups were executed within 3 to 5 months, and their pancreatic tissues were observed
by macrography and under microscopy. Meanwhile, the rats in the control group (group
C) were executed at 5 months. Immunohistochemistry was used to assay the expression
of MGMT, ERCC1, hMSH2, and hMLH1.

Results

The incidence of pancreatic cancer in group A within 3 to 5 months was 48.7% (18/37),
including 1 case of fibrosarcoma. The incidence of pancreatic cancer in group B was
33.3% (12/36), including 1 case of fibrosarcoma. The mean of maximal diameters of
tumors in group A was higher than that in group B (P <0.05). No pathological changes were found in pancreas of group C and other main
organs (except pancreas) of group A and group B. No statistical differences were found
among the positive rates of MGMT, ERCC1, hMSH2, and hMLH1 in ductal adenocarcinoma and non-cancerous pancreatic tissues of group A (P >0.05). The positive rates of MGMT, ERCC1, hMSH2, and hMLH1 were significantly lower in ductal adenocarcinoma than those in non-cancerous tissues
of group B (P ≤0.05). All pancreas of group C had positive expression of MGMT, ERCC1, hMSH2, and hMLH1 and two cases of fibrosarcoma showed a negative expression.

Conclusions

DMBA, directly implanted into the parenchyma of pancreas, creates an ideal pancreatic
cancer model within a short time. TSA might restrain DNA damage related to the genesis
and growth of pancreatic cancer in rats. The DNA-repair proteins, including MGMT,
ERCC1, hMSH2, and hMLH1, might play an important role in the genesis of pancreatic cancer induced by DMBA
in rats.

Keywords:

Background

Pancreatic cancer is a solid malignancy characterized by its rapid growth and propensity
to invade adjacent organs and metastasize. Worldwide, pancreatic cancer causes approximately
213,000 deaths each year. The 1-year survival rate is around 20% and 5-year survival
rate is less than 5% in spite of aggressive therapies [1]. Within the last two decades, research has shown that pancreatic cancer is fundamentally
a genetic disease caused by inherited germline and acquired somatic mutations in cancer-associated
genes, and more and more investigation of molecular pathogenesis has been used in
the diagnosis and treatment of pancreatic cancer. To build useful models studying
the pathological molecular mechanisms of pancreatic cancer, Rivera et al. directly
implanted dimethylbenzanthracene (DMBA) into the parenchyma of the rat pancreas and
found a pancreatic cancer incidence of 39% within 10 months [2]; Bockman et al. reported similar studies [3].

Trichostatin A (TSA) is a histone deacetylase inhibitor with a broad spectrum of epigenetic
activities. It can up-regulate the expression of several genes and restrain other
genes’ expression, thus intervening in the genesis and development of tumors. In vivo or in vitro experiments have confirmed that TSA could restrain the genesis of some tumors and
control tumor progression by restraining tumor angiogenesis and changing the tumor
microenvironment [4]. Some studies have shown that TSA acts as a tumor suppressor in human pancreatic
cancer cell lines [5,6].

The DNA mismatch repair (MMR) system is an inbuilt security system that can repair
DNA mismatch in human cells, and plays an important role in retaining the integrality
and stability of genes. The main MMR genes are hMSH1-6, hMLH1-5 and others, and the methylation of MMR genes and/or the loss of expression of their
proteins plays an important role in malignant tumorigenesis [7-11]. O6-methylguanine DNA methyltransferases (MGMT) is a high-performance DNA-repair enzyme,
which can protect cells from alkylating agent damage and prevent cell carcinogenesis
[11-16]. Excision repair cross-complementing gene 1 (ERCC1) is a member of the exonuclease repair enzyme family, and its low expression is always
related with elevated cancer incidence while its high expression is always related
with resistance to platinum drugs [17-21].

Since no studies have examined the expression levels of DNA-repair proteins (MGMT,
ERCC1, hMSH2, and hMLH1) in pancreatic cancer induced by DMBA and non-cancerous pancreatic cancer tissues
in rats, little is known about the effects of MGMT, ERCC1, hMSH2, and hMLH1 on rat pancreatic cancer induced by DMBA. In this study, DMBA was directly implanted
into the parenchyma of the pancreas of rats to establish a pancreatic cancer model,
and TSA injection was given to establish the intervention group. The expression levels
of MGMT, ERCC1, hMSH2, and hMLH1 in pancreatic cancer and non-cancerous pancreatic tissues was detected and their
effect on the process of inducing cancer by DMBA was assessed.

Methods

Animal model

Ninety Sprague–Dawley (SD) rats (no sex limit), weighing between 150 and 200 g were
used. These rats were randomly divided into three groups: 40 in the pancreatic cancer
model group (group A), 40 in the TSA intervention group (group B), and 10 in the control
group (group C). The rats were treated with preoperative fasting for 24 hours (no
water ban), and 2% amyl-barbital was injected into the abdomen under anesthesia. The
rats’ abdomens and parenchyma were then dissected (1 mm) and DMBA (9 mg) was directly
implanted into the parenchyma of the pancreas in groups A and B, followed by suturing.
The rats were raised in common conditions after operation, and rats in group B were
injected with 1 mL TSA (1 μg/mL) weekly through the abdomen. Except for natural death,
the rats were executed randomly in the third month (7 rats in group A and 6 rats in
group B), in the fourth month (10 rats in both groups A and B), and in the fifth month
(20 rats in both groups A and B) after operation. Rats in group C, which were treated
without DMBA implantation and treated in the same condition as group A, were executed
in the fifth month after operation.

The design of this study was approved by the medical ethics commitee of the Second
Hospital of Yueyang City.

Macrography and pathological observation

The livers, gallbladder, stomach, intestine, and lung of rats in groups A and B were
observed by macrography. After which the whole pancreatic tissues and some tissues
from the liver, gallbladder, stomach, intestines, and lungs of rats were put into
4% formaldehyde for 16 to 18 hours. Conventional paraffin-embedded sections were made
with these specimens. Finally the sections were dyed by hematoxylin and eosin staining,
and observed under microscopy.

Immunohistochemical staining of EnVision™

Immunostaining was conducted by use of the ready-to-use, peroxidase-based EnVision™
Detection kit (Dako Laboratories, CA, USA) according to the user manual. Four-micrometer-thick
sections were cut from routinely paraffin-embedded tissues.

Statistical analysis

Data was analyzed by using the statistical package for the Social Sciences Version
13.0 (SPSS 13.0). All the data were analyzed by using χ2 test, rank-sum test, and Fisher’s exact test.

Results

Macrography

The incidence of pancreatic tumors in groups A and B are shown in Table 1; the incidence of tumors in group A was higher than that in group B (P >0.05). Both groups A and B had one case of fibrosarcoma that developed liver metastasis
and epiploon metastasis. The distribution of diameter of tumor mass in group A was
0.5–1.0 cm (7 cases), 1.0–2.0 cm (10 cases), and >2.0 cm (1 case); and the distribution
of diameter of tumor mass in group B was 0.5–1.0 cm (9 cases), 1.0–2.0 cm (2 cases),
and >2.0 cm (1 case). The mean of maximal diameter of tumors in group A was higher
than that in group B (P <0.05). No pathological changes were found by macrography in pancreas of group C
and other main organs (except pancreas) of groups A and B.

Table 1.Incidences of pancreatic tumors in groups A and B (case number ratio (%))

Pathological observation

Pathological results of pancreatic tumors in groups A and B are shown in Table 2 and Figure 1A. Both non-cancerous pancreatic tissues and peritumoral pancreatic tissues in groups
A and B showed hyperplasia to atypical-hyperplasia. Non-cancerous pancreatic tissues
in group A which showed mild atypical-hyperplasia were found in 5 cases (26.3%) and
moderately to severely atypical-hyperplasia in 10 cases (52.6%). The same tissues
were found in group B in 10 cases (41.6%) and 8 cases (33.3%), respectively; therefore,
no statistical differences were found in the two groups (P >0.05). No pathological changes were found by microscopy in pancreas of group C and
other main organs (except pancreas) of groups A and B.

Table 2.Pathological types of pancreatic tumors in groups A and B (case number)

The positive rates of MGMT, ERCC1, hMSH2, and hMLH1 (Figure 2) were significantly lower in ductal adenocarcinoma than those in non-cancerous pancreatic
tissues in group A + group B (P <0.01 or P <0.05). No statistical differences were found among the positive rates of MGMT, ERCC1, hMSH2, and hMLH1 in ductal adenocarcinoma and non-cancerous pancreatic tissues of group A (P >0.05). The positive rates of MGMT, ERCC1, hMSH2, and hMLH1 were significantly lower in ductal adenocarcinoma than those in non-cancerous tissues
of group B (P <0.05). The ductal epithelium of non-cancerous pancreas which had negative expression
of MGMT, ERCC1, hMSH2, and hMLH1 in groups A and B all showed moderately or severe atypical-hyperplasia. The fibrosarcoma
had negative expression of MGMT, ERCC1, hMSH2, and hMLH1, while pancreas of group C had positive expression of MGMT, ERCC1, hMSH2, and hMLH1 (Table 3). Expression of MGMT, ERCC1, hMSH2, and hMLH1 had no obvious correlation with the size of tumor mass and differentiation degree
of ductal adenocarcinoma (P >0.05).

Discussion

Establishing of a pancreatic cancer model can be achieved through three kinds of methods
[24-27]: 1) exposing canine animal to carcinogen, 2) activating the oncogenes of transgenic
mice, and 3) transplanting the xenogenic pancreatic cancer tissues to athymic mouse.
Rivera et al. directly implanted DMBA into the parenchyma of rat pancreas to establish
a pancreatic cancer model of rats and the incidence of cancer of SD rats within 10
months was 39% [2]. Since then, a series of mouse and rat pancreatic cancer models using DMBA have been
established [28-32]. TSA can increase intracellular histone levels and up-regulate the expression of
several genes. Some experiments have confirmed that TSA can restrain the genesis of
some tumors by restraining angiogenesis, inhibiting proliferative activity, and promoting
apoptosis of tumor cells [33-37]. After we directly implanted a major dose of DMBA (9 mg) into the pancreas parenchyma
of SD rats, the incidence of cancer in group A within 3 to 5 months was 48.7%, and
that in group B was 33.3%; their pathological types were the same as those of human
pancreatic ductal adenocarcinoma, except for two cases of fibrosarcoma. The incidence
of cancer in group A was higher than that in group B, but the difference had no statistical
significance (P >0.05). The mean of maximal diameter of tumors in group A was higher than that in
group B (P <0.05). Our SD rat model of pancreatic cancer had some merits: 1) the period of tumor
formation was short and the incidence of cancer was high; 2) the pathological type
was mainly the same as human pancreatic ductal adenocarcinoma; 3) no pathological
changes were found in main organs (except pancreas); 4) the inhibitive effect on carcinogenesis
and growth of TSA was obvious; and 5) the cost was low.

MGMT is a high-performance DNA-repair enzyme that can protect cells from alkylating
agent damage and can prevent cell carcinogenesis and death. The MGMT gene is located in 10q26 and encodes 207 amino acids’ proteins [7-11]. Normal cells all have MGMT expression, while some malignant tumors will lose MGMT
expression which will induce the damage of DNA repair and the carcinogenesis of cells
[7-11,38,39]. ERCC1 is a member of the exonuclease repair enzyme family and its low expression is always
related to elevated cancer incidence, while its high expression is always related
to resistance to platinum drugs [12-16]. Recent studies have confirmed that ERCC1 is the key enzyme of the DNA repair induced by cisplatin and it has been shown that
ERCC1 expression of some malignant tumors played an important role in guiding chemotherapy
[17-21,37]. The hMSH2 gene is located in 2P16 and is the first separated MMR. It can repair DNA mismatch and retain the integrality
and stability of genes. Many recent papers have reported that the loss of hMSH2 protein expression was crucial to the genesis and progression of malignant tumors
[7-11,40-42]. hMLH1 is also a type of MMR which can also inhibit carcinogenesis by repairing DNA mismatching.
Mutation of the hMLH1 gene will induce the genesis of many malignant tumors [7-11,41].

Conclusions

Our data have shown that the positive rates of MGMT, ERCC1, hMSH2, and hMLH1 were significantly lower in pancreatic ductal adenocarcinoma than in non-cancerous
pancreatic tissues of rats, and the ductal epithelia of non-cancerous pancreas which
had negative expression of MGMT, ERCC1, hMSH2, and hMLH1 all shown atypical-hyperplasia. The results show that there was loss expression of
MGMT, ERCC1, hMSH2, and hMLH1 in the course of genesis of pancreatic cancer induced by DMBA in rats, which might
be the mechanism of carcinogenesis by DMBA. Therefore, testing the expression of MGMT,
ERCC1, hMSH2, and hMLH1 in pancreatic cancer might play an important role in guiding the treatment of human
pancreatic cancer.

Competing interests

The authors report no competing interests. No benefits in any form have been received
or will be received from a commercial party related directly or indirectly to the
subject of this article.

Authors' contributions

TXG did most of the experiments and data acquisition, TXG and YZ participated in the
design of experiments, interpretation of data, and writing of the manuscript. LY and
XYM participated the experiments and writing. All authors read and approved the final
manuscript.